The determination of the presence of analogs/isoforms of endogenous compounds is a growing field in clinical and research medical science. Such diagnostic methods are useful in, e.g., the treatment of diabetes, the treatment of metabolic syndrome, or doping controls.
Current methods for assessing the risk for or diagnosing pathologic conditions such as diabetes often rely on a diagnosis by attrition, a process of elimination or by invasive surgery or biopsies. In certain diseases, such as metabolic disease or doping, the methods by which an objective diagnosis may be made are often indirect, cumbersome, time-consuming and costly, see, e.g., LC-MS/MS.
During the past decade, several therapeutic peptides and proteins have been developed for the treatment of patients with diabetes mellitus and metabolic syndrome. After the introduction of recombinant human insulin in the 1980ies, insulin analogs have been approved for treatment that vary in their amino acid sequence, and in consequence have a different pharmacokinetic profile. Next to short acting insulin analogs (insulin Lispro (Eli Lilly & Co, Indianapolis, Ind.), insulin Aspart (Novo Nordisk Pharmaceuticals, Baulkham Hills, Australia), and insulin Glulisine (Aventis, Bridgewater, NL)) for prandial insulin substitution, longer acting basal insulin analogs (insulin Glargine (Sanofis-Aventis, Frankfurt am Main, Del.) and insulin Detemir (Novo Nordisk Pharmaceuticals, Baulkham Hills, AU) provide the insulin coverage for the remaining portions of the day not covered by short-acting insulin analogs. As combinations of these analogs provide several clinical advantages towards regular human insulin (e.g. less risk of hypoglycemia or no requirement of an injection to meal interval), they are nowadays widespreadly used worldwide. In some clinical situations (e.g. hypoglycemia) and also in many scientific projects, it has become desirable to be able to specifically measure the concentrations of these analogs in the blood of patients and thus multiple efforts have been undertaken to develop analog-specific immunoassays for these determinations. However, as of today only one assay that is specific for insulin Lispro is commercially available after many years of developmental efforts (Millipore, St. Charles, US).
Indirect assessments are only possible by means of cross-reactive insulin assays, which, however, cannot distinguish between regular human insulin and/or several analogs thereof.
For example, Owen and Roberts (2004) describe cross-reactivity of three recombinant insulin analogs with five commercial insulin immunoassays.
Lindström et al. (2002) disclose a double-antibody technique to describe the pharmacokinetics of the rapid-acting insulin analogs Aspart and Lispro. The antibody used recognizes human insulin but does not cross-react or specifically distinguish the two insulin analogs. The combination of such an immuno assay with a second immuno assay using an antibody that cross-reacts with all three forms allows a determination of the ratio between insulin and its two analogs, but these tests do not allow a differentiation between the two analogs or the determination of other basal and rapid-acting insulin analogs and insulin.
Bowsher et al. (1999) describes a radio immuno assay (RIA) for the specific determination of insulin Lispro that does not show interference from insulin, proinsuline, or C-peptide and has interassay CVs of 2.6-13.4%. However, the antiserum does not allow a differentiation between further analogs of insulin and insulin.
Andersen et al. (2000) describe an insulin immunoassay specific for the rapid-acting insulin analog insulin Aspartat. Nevertheless, the assay does not distinguish further analogs of insulin and is not commercially available.
Cao et al. (2001) describe an immunoassay for the determination of carboxyl-terminal B-chain analogues of human insulin that can quantify human insulin, proinsuline, despentapeptide insulin, procine insulin and insulin Lispro with comparable cross-reactivity, i.e. it shows cross-reactions between the listed analogs and is predicted to show cross reaction to further analogs.
An Elecsys® assay of free insulin determination and the absence of cross-reactivity with insulin Lispro is disclosed in Sapin et al. (Clin. Chim. Acta 2001). However, the assay cross-reacts with further insulin analogs such as pig insulin.
Up to date, the common presence or concentration of further human insulin analogs and endogenous human insulin in a patient's sample (for example, a blood sample) can only be determined in the common presence of the insulin forms by means of sophisticated and work-intense analytical methods with unreasonable requirements as regards costs and human resources (e.g. liquid chromatography-tandem mass spectrometry (LC-MS/MS)).
Thevis et al. (2005) describe a qualitative determination of synthetic analogs of insulin in human plasma by immuno affinity purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for doping control purposes. However, this method requires extensive sample preparation prior and subsequent to the chromatographic step.
Cao et al. (2006) report of HPLC-MS method development for quantification of insulin and its analogs and for identification of the source of discrepancy among immunoassay methods. Similar to the method described in Thevis et al., the HPLC-MS method requires extensive sample preparation.
Electrophoresis is a well-established technology for separating particles based on the migration of charged particles under the influence of a direct electric current. Several different operation modes such as isoelectric focusing (IEF), zone electrophoresis (ZE) and isotachophoresis (ITP) have been developed as variants of the above separation principle and are generally known to those of skill in the art.
IEF (isoelectric focusing), one of the above general operation modes of electrophoresis, including free flow electrophoresis, is a technique commonly employed, e.g., in protein characterization as a mechanism to determine a protein's isoelectric point (see, e.g., Analytical Biochemistry, Addison Wesley Longman Limited-Third Edition, 1998) or to separate analytes according to their isoelectric point (pI). IEF is discussed in various texts such as Isoelectric Focusing by P. G. Righetti and J. W. Drysdale (North Holland Publ., Amsterdam, and American Elsevier Publ., New York, 1976). Zone electrophoresis (ZE) is another alternative operation mode based on the difference between the electrophoretic mobility value of the particles to be separated and the charged species of the separation medium employed.
WO 2007/147862, which is herewith incorporated in its entirety, discloses combinations of ZE and IEF in FFE.
Isotachophoresis (ITP) is a more recent variant of electrophoresis wherein the separation is carried out in a discontinuous buffer system. Sample material to be separated is inserted between a “leading electrolyte” and a “terminating electrolyte”, the characteristics of buffers being that the leader will comprise ions having a net electrophoretic mobility higher than those of the sample ions, while the terminator must comprise ions having a net electrophoretic mobility lower than those of the sample ions. In such a system, sample components sort themselves from leader to terminator in accordance with their decreasing mobilities in a complex pattern governed by the so-called Kohlrausch regulating function. The process has been described in the art, for instance, in Bier and Allgyer, Electrokinetic Separation Methods 443-69 (Elsevier/North-Holland 1979).
International patent application PCT/EP2007/061840, which is incorporated herein by reference in its entirety, refers to media combinations for enhanced free flow ITP.
Among electrophoretic technologies, free flow electrophoresis (FFE) is one of the most promising [Krivanova L. & Bocek P. (1998), “Continuous free-flow electrophoresis”, Electrophoresis 19: 1064-1074]. FFE is a technology wherein the separation of the analytes occurs in a carrier-free medium, i.e., a liquid (aqueous) medium in the absence of a stationary phase (or solid support material) to minimize sample loss by adsorption. FFE is often referred to as carrier-less deflection electrophoresis or matrix-free deflection electrophoresis.
A particular FFE technique referred to as interval FFE is disclosed, for example, in U.S. Pat. No. 6,328,868. In this patent, the sample and separation medium are both introduced into an electrophoresis chamber, and the analytes in the sample are separated using an electrophoresis mode such as ZE, IEF or ITP, and are finally expelled from the chamber through fractionation outlets. Embodiments of the '868 patent describe the separation media and sample movement to be unidirectional, traveling from the inlet end towards the outlet end of the chamber, with an effective voltage applied causing electrophoretic migration to occur while the sample and media are not being fluidically driven from the inlet end towards the outlet end, in contrast to the technique commonly used in the art wherein the sample and media pass through the apparatus while being separated in an electrical field (commonly referred to as continuous FFE).
Another particular FFE technique referred to as cyclic or cyclic interval mode has been described in International application WO 2008/025806, hereby incorporated by reference in its entirety. In sum, the cyclic interval mode is characterized by at least one, and possible multiple reversals of the bulk flow direction while the sample is being held in the electrophoretic field between the elongated electrodes. In contrast to static interval mode, the sample is constantly in motion thereby allowing higher field strength and thus better (or faster) separation. Additionally, by reversing the bulk flow of the sample between the elongated electrodes, the residence time of the analytes in the electrical field can be increased considerably, thereby offering increased separation time and/or higher separation efficiency and better resolution. The reversal of the bulk flow into either direction parallel to the elongated electrodes (termed a cycle) can be repeated for as often as needed in the specific situation, although practical reasons and the desire to obtain a separation in a short time will typically limit the number of cycles carried out in this mode.
International patent application WO 2002/50524 A and U.S. patent application 2004/050698 and International patent application PCT/EP2007/061840, which are incorporated herein by reference in their entireties, disclose an FFE apparatus useful for FFE separations as described above.
A number of separation media for the separation of analytes such as bioparticles and biopolymers are known in the art. For example, the book “Free-flow Electrophoresis”, published by K. Hannig and K. H. Heidrich, (ISBN 3-921956-88-9) reports a list of separation media suitable for FFE and in particular for free flow ZE (FF-ZE).
U.S. Pat. No. 5,447,612 discloses another separation medium which is a pH buffering system for separating analytes by isoelectric focusing by forming functionally stable pre-cast narrow pH zone gradients in free solution. It employs buffering components in complementary buffer pairs.
Binary buffer systems referred to herein as “NB buffer systems” are disclosed in detail in International patent application PCT/EP2008/050597, which is incorporated herein by reference in its entirety. The buffer system comprises at least one buffer acid and at least one buffer base, with the proviso that the pKa value of the buffer acid must be higher than the pH of the separation buffer medium (SBM) and the pKa of the buffer base is lower than the pH of the SBM. Put another way, the pKa of the buffer acid will be higher than the pKa of the buffer base.
U.S. co-pending provisional applications U.S. Ser. Nos. 60/945,246 and 60/987,208, which are incorporated herein by reference in their entireties, refer to volatile buffer systems suitable for FFE. The volatile buffer systems offer the advantage that they can be easily removed subsequent to a FFE step and prior to a downstream analysis such as MS, or that they do not disturb a downstream analysis.